Microfluidic reaction apparatus for high throughput screening

ABSTRACT

An SBS-formatted microfluidic device where the geometry of the plate defines an array of interrogation areas, and where each interrogation area encompasses at least one reaction site.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/680,541 filed Feb. 28, 2007, which application claims priority toU.S. Provisional Application Ser. No. 60/777,972 filed Feb. 28, 2006,U.S. Provisional Application Ser. No. 60/849,223 filed Oct. 4, 2006, andU.S. Provisional Application Ser. No. 60/881,627 filed Jan. 19, 2007, ofwhich the entire contents of all the applications are hereinincorporated by reference for all purposes.

In addition, embodiments of microfluidic structures and materials usedin the devices of the present invention are described in U.S. patentapplication Ser. No. 11/006,522, filed Dec. 6, 2004, and published Sep.22, 2005 as U.S. Pat. Pub. No. 20050205005, with the title “MICROFLUIDICPROTEIN CRYSTALLOGRAPHY”, the entire contents of which are herebyincorporated by reference for all purposes. Applications of the methodsand devices of the present invention are also related to the inventionsdescribed in PCT application PCT/US01/44869, filed Nov. 16, 2001, andentitled “CELL ASSAYS AND HIGH THROUGHPUT SCREENING”; U.S. patentapplication Ser. No. 10/116,761, filed Apr. 3, 2002, and published Jan.9, 2003 as U.S. Pat. Pub. No. 20030008411, with the title “COMBINATORIALSYNTHESIS SYSTEM”; U.S. patent application Ser. No. 10/416,418, filedNov. 16, 2001, and published Jun. 17, 2004 as U.S. Pat. Pub. No.20040115838, with the title “APPARATUS AND METHODS FOR CONDUCTING ASSAYSAND HIGH THROUGHPUT SCREENING”; and U.S. patent application Ser. No.10/118,466, filed Apr. 5, 2002, and issued Nov. 1, 2005 as U.S. Pat. No.6,960,437, with the title “NUCLEIC ACID AMPLIFICATION UTILIZINGMICROFLUIDIC DEVICES”, which are also hereby incorporated by referencefor all purposes.

Additional information relating to the formation of microfabricatedfluidic devices utilizing elastomer materials are described generally inU.S. Pat. No. 6,408,878, filed Feb. 28, 2001, entitled “MICROFLUIDICELASTOMERIC VALVE AND PUMP SYSTEMS”; U.S. Pat. No. 6,899,137, filed Apr.6, 2001, entitled “MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS”;U.S. patent application Ser. No. 09/724,784 filed Nov. 28, 2000,entitled “MICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS”; and Ser.No. 09/605,520, file Jun. 27, 2000, entitled “MICROFABRICATEDELASTOMERIC VALVE AND PUMP SYSTEMS.” These patents and patentapplications are also hereby incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Microtiter plates have become a standard tool in chemistry, biology andmedical laboratories. The plates are typically flat glass or plastictrays in which an array of circular reagent wells are formed. Each wellcan typically hold between from a few microliters to hundreds ofmicroliters of fluid reagents and samples, which may be loaded into thewells with automated delivery equipment. Plate readers are used todetect biological, chemical and/or physical events in the fluids placedin each well.

As the fields of combinatorial chemistry and high throughput screeninghave grown, so has equipment and laboratory instrumentation that hasbeen designed to fill, manipulate and read microtiter plates.Unfortunately, the equipment makers made little effort develop systemsthat were cross-compatible with the systems of other manufacturers. Bythe mid-1990s, the Society for Biomolecular Screening (SBS) formed astandards group to address these cross-compatibility problems. A finalset of standards was published by SBS and the American NationalStandards Institute 2003.

These standards define the overall dimensions of a compliant microtiterplate, as well as the diameter, depth and spacing of the reagent wellsin the plate. The plates may include 96, 384, 1536, etc., wells arrangedin a 2:3 rectangular matrix. While some manufacturers have made platespacking even larger numbers of reagent wells into the dimensions of anSBS-formatted plate, the small-sizes of the wells can make filling andreading the plates more difficult. Thus, there is a need for devices,systems and methods that can rapidly and accurately deliver smallvolumes of samples and reagents to reaction sites in high throughputmicrotiter plates. There is also a need for devices, systems and methodsthat provide monitoring, detecting and reading of reactions performed atthe reaction sites of such microtiter plates. There is also a need todesign such microtiter plates to SBS compatible standards, so they cantake advantage of the large amount of SBS-formatted equipment andinstrumentation that is currently in use. These and other needs areaddressed by the present invention.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention include SBS-formatted microfluidic devicesthat include a plate having an upper surface and a lower surface, wherethe geometry of the plate defines an array of interrogation areas. Theperimeter of each interrogation area defines a region on the plate whereSBS-compatible equipment can measure and/or influence reactions takingplace on the plate. Typically, the interrogation areas have the samesize and position as the reaction wells in a conventional SBS-formattedmicrotiter plate. Each interrogation area may encompass one or morereaction sites. These reaction sites may be sealable chambers in whichreactants, samples, etc., may be brought into contact and reacted. Thereaction sites may be isolated from each other, allowing multipledifferent reactions to take place at the same time in the sameinterrogation area. The reaction sites may lie beside each other withinan interrogation area and be defined by the presence of one or morereactants or analytes that have been deposited upon a specific area onthe substrate surface of the plate.

The devices may also include one or more elastomeric layers, generallypositioned upon a rigid substrate surface, wherein a plurality ofchannels is disposed within the elastomeric layers or is defined at theinterface between the elastomeric layers or at the interface of anelastomeric layer and the rigid substrate surface. In certainembodiments, a first elastomeric layer in contact with the upper surfaceof the plate, where the first elastomeric layer comprises a network ofmicrofluidic flow channels formed therein in fluid communication with anarray of reaction sites. The devices may further include a secondelastomeric layer in contact with the first elastomeric layer, where thesecond elastomeric layer includes a network of control channels formedtherein, configured to control the flow of fluids within the flowchannels.

It should be noted that all reference to relative positions of the plateand elastomeric layers, such as “upper surface” and “lower surface” areprovided solely for the purpose of conceptual convenience, and are notmeant to restrict the invention to such arrangements. The devices of theinvention may be made with the plate either above or below theelastomeric layers or in any other arrangement such that the elementsprovide a functioning microfluidic system.

Embodiments of the invention may also include systems of SBS-formattedmicrofluidic devices that can process at least one flowable reagent. Thereagent may include any liquid, and may include a dye or other reagentdetectable by optical or other sensors. The systems may also becompatible with machines used to read the device or to monitor areaction performed upon the device. The machines may be SBS compatibleand able to accept, use, manipulate, and read an SBS-formatted plate. Inaddition, the systems may include machines for dispensing and/or pumpingfluids into the microfluidic devices. These dispensing and pumpingmachines may also be SBS compatible and able to accept an SBS-formattedplate.

Embodiments may still further include systems to manipulate, read and/ormonitor one or more reactions performed upon or within a SBS-formattedmicrofluidic device. The systems may include a SBS-formattedmicrofluidic device and a machine for reading, monitoring and/ormanipulating a reaction performed upon or within the SBS-formattedmicrofluidic device.

Embodiments of the invention may still also include processes forconducting microfluidic high throughput sample reaction measurements.The processes may include the step of providing a microfluidic testingdevice comprising a reaction plate that has an array of reaction sites,such as within one or more wells. In certain embodiments, each welldefines a single reaction site for the performance of a single reaction.In additional embodiments each well has at least two reaction sites,allowing two or more reactions to be performed in a single well. Thereaction sites may be physically defined by a reaction chamber. Thereaction chambers may be coupled to reagent flow channels and sampleflow channels that are formed in a first elastomeric layer adjacent tothe reaction plate. The processes may also include loading a set ofreagents into an array of reagent reservoirs, and loading a set ofsamples into an array of sample reservoirs of the microfluidic testingdevice. Each of the reagent reservoirs is fluidly coupled to one of thereagent flow channels, and each of the sample reservoirs is fluidlycoupled to one of the sample flow channels. The processes may furtherinclude transporting at least one of the reagents through at least oneof the reagent flow channels to a designated reaction chamber and atleast one of the samples through at least one of the sample flowchannels to the designated reaction chamber. The control channelscontrol the flow of the reagent and the sample through the flow channelsto the designated reaction chamber, and are formed in a secondelastomeric layer adjacent to the first elastomeric layer. The processesmay still further include the step of measuring a reaction response fromthe reagent and the sample in the designated reaction chamber.

In certain embodiments, two or more reaction sites may be present withina single interrogation site. The interrogation site may be defined, forexample, as an imaging area upon an SBS-formatted chip, which is imagedby a single lens at a single set position. This interrogation area mayhave defined within it any number of reaction sites. Any or all of thesereaction sites may be imaged simultaneously by a lens positioned abovethe reaction site. A single image of a single interrogation areacaptured by a single lens may contain information pertaining to anynumber of reaction sites within the imaged interrogation area. Theinformation associated with each reaction site may be parsed anddifferentiated from the information associated with any other reactionsite in a number of ways.

For example, a device having four reaction sites within a singlereaction area (e.g., FIG. 2) may be set-up such that each reaction ateach of the four reactions sites produces a different colored signalsignifying completion of a reaction (e.g., green, red, yellow, and bluecolored signals). The optical image captured by a single lens may beoptically or electronically split up into its component colors toprovide for separate signals. These signals may be processed eitherquantitatively (to determine signal strength at a particular reactionsite) or qualitatively (to determine the presence or absence of areaction). Alternatively, with a single color reaction, the reactioncould be set up to sequentially run a reaction at each of the fourreaction sites. One reaction per interrogation area may be performed ata time, and then be read. That reaction chamber may then be bleached orwashed out and a second reaction may be performed at the second reactionsite, and so forth, until all four reactions had been completed andread.

Embodiments of the invention still also include systems having lens tofocus light on the reaction sites in interrogation areas.

Additional embodiments and features are set forth in part in thedescription that follows, and in part will become apparent to thoseskilled in the art upon examination of the specification or may belearned by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective view of a SBS-formatted microfluidic plateaccording to embodiments of the invention.

FIG. 1B is a plan view of an SBS-formatted microfluidic plate accordingto embodiments of the invention.

FIG. 2 is a group of 2×2 reaction sites that make up a portion of areaction site array according to embodiments of the invention.

FIGS. 3A-B are illustrations of optical arrangements that may be usedwith spectral measurement instrumentation according to embodiments ofthe invention.

FIG. 4 is an illustration of a portion a network of control and flowchannels that supply fluids to reaction sites according to embodimentsof the invention.

FIG. 5 is an exploded view of the SBS-formatted microfluidic plateaccording to embodiments of the invention.

FIGS. 6A-C are schematic views of SBS-formatted microtiter plates for96, 384 and 1536 reagent wells.

FIG. 7 shows an enlarged view of one embodiment of a chip holder inaccordance with the present invention.

FIGS. 8A-D are simplified schematic diagrams illustrating positivedisplacement cross-injection (PCI) dispensing.

FIGS. 9A-C are diagrams of exemplary carry-on reaction cell designs.

FIG. 10 is an embodiment of a carry-on (or pig mix) reaction celldesign.

FIG. 11 is a diagram of a portion of a 32×32 carry-on reaction cellmatrix.

FIGS. 12A-H are micrographs showing mixing in a portion of a 32×32carry-on reaction cell matrix.

FIG. 13 is a diagram of an exemplary carry-on mixing and meteringchannel with multiple slug injection segments.

DETAILED DESCRIPTION OF THE INVENTION

Devices, systems and processes are described that integrate microfluidicsample delivery and reaction technology with existing laboratory devicesand methods that may be used for performing high throughput testing ofchemical and biological samples, and for carrying out chemical andbiological reactions, especially those employing small liquid volumes.These devices and systems may include fluorescent plate readers,real-time PCR machines, robotic plate handlers, and pipetting robots,and devices designed to load, manipulate and read elastomericmicrofluidic devices, among other types of equipment. The devices,systems and methods of the invention are compatible with one or morestandards that allow them to easily interface with existing laboratoryequipment. For example, the components may conform to the widely adoptedSBS format for automated laboratory instrumentation.

The devices and systems of the invention may include arrays of reactionreservoirs that are formatted for compatibility with automated reactantloading equipment (e.g., pipetting robots) that already exist and are incommon usage in laboratories and manufacturing facilities. The devicesand systems may also include arrays of reaction product reservoirs thatreceive reaction products from reactions chambers where selectedcombinations of the reactants were combined. The reaction productreservoirs may also be formatted for compatibility with pre-existingautomated sample extraction equipment to transport reaction productsamples to analysis equipment, storage containers, etc.

Integrating microfluidic sample delivery technology with high throughputtesting equipment combines advantages from both fields. Microfluidicsystems have fewer moving parts and simpler operational logistics thanrobotic fluid delivery systems. In general, the microfluidic systemscost less to manufacture and require less maintenance and repair. Inaddition, microfluidic systems can be manufactured with smaller sizedconduits and chambers, allowing them to deliver smaller volumes ofsamples, reagents, etc., than practicable with, for example, pipettingrobots. This can reduce the costs and waste products generated for largescreening studies involving thousands or more combinations of reagentsand samples. The small volumes can also make screening and combinatorialstudies practical when only a small amount of a sample is available.

Smaller component dimensions also permit more densely packedarrangements of the reaction sites. For example, two, four, eight, ormore microfluidic reaction chambers (each defining a reaction site) maybe packed into the interrogation area of a single site for astandardized high throughput screening device. This can allow themicrofluidic device to achieve a twofold, fourfold, eightfold, or more,increase in the throughput rate using an existing screening device.

Microfluidic Device Overview

FIG. 1A shows a microfluidics plate 100 that includes a reaction sitearray 102 positioned between two reservoir arrays 110 a-b. Each reactionsite 106 may have a surface area from about 0.5 mm² to about 315 mm²,and may have a volume of about 1 nL to about 1000 nL each, and may beconfigured to make the array 102 compatible with an SBS formattedmicroplate reader. As FIG. 1B shows for this example, there are 192reaction sites 106 in the array 102 arranged in 12 rows by 16 columns.This array of reaction sites 106 overlaps an equal number ofinterrogation areas 108 in the SBS-formatted interrogation area array.The 384 well SBS-formatted plate 100, has a 16×24 array of interrogationareas 108. The reaction sites 106 overlap with a subset of theinterrogation area array in the middle of the plate 100. In thisarrangement, space for four additional SBS-formatted columns exists oneither side of the 16 columns of reaction sites 106, and space for 2additional SBS-formatted rows exists on either side of the 12 rows ofreaction sites 106.

As FIG. 1B shows, some of the space on the sides of the reaction sitearray 102 may be used for the reservoir arrays 110 a-b. In the exampleshown, a 16 row by 3 column reservoir array 110 a is positioned on oneside of the array 102, and an 8 row by 3 column reservoir array 110 b ispositioned on the opposed side of the array 102. The configuration ofthe individual reservoirs in the arrays 110 a-b may also be formattedaccording to the SBS standard. Thus, automated filling and extractingequipment, such as a pipetting robot, may be programmed to fill orremove fluids from the reservoirs in the same manner as the reagentwells of a conventional, SBS-formatted, microtiter plate.

The reservoir arrays 110 a-b may be filled with reagents, reactants,samples, reaction products, etc., in any configuration. For example, oneof the reservoir arrays (e.g., array 110 a) may be filed with reagents,and the second array 110 b may be filled with samples. Individualcombinations of the reagents and samples may be combined at the reactionsites in array 102 and monitored by SBS-formatted measuring equipmentthat defines an interrogation area 108 each of the reaction sites. Inanother example, one of the reservoir arrays (e.g., array 110 a) mayhold reactants, and the second array 110 b may receive reaction productsfrom the reactions that take place in the reaction sites of array 102.The reservoir arrays 110 a-b may also be subdivided into sub-arraysand/or at the level of individual reservoirs into different groupings offluids.

The space around the perimeter of plate 100 occupied by the reservoirarrays 110 a-b, channels that supply fluid to the array 102, pumpingequipment, etc., may take up space that would otherwise be occupied bywells in a conventional microtiter plate. This may result in fewerinterrogation areas 108 in plate 100 than wells in a conventional plate(which can define the size and position of the interrogation areas 108).For example, the reaction site array 102 of plate 100 shown in FIG. 1Bencompasses 192 interrogation areas 108, which is half the number ofwells in a conventional SBS-formatted 384 well microtiter plate. Whenthe reaction sites 106 in array 102 overlap the interrogation areas 108in a 1:1 ratio, there are half the number of reaction sites (i.e., 192)as available in a conventional SBS-formatted microtiter plate (i.e., 384wells). But packing more reaction sites within the perimeter of eachinterrogation area 108 can quickly increase total number of reactionssites beyond what is possible with a conventional microtiter plate.

For example, if just 2 reaction sites 106 were packed into eachinterrogation area 108 (i.e., a 2:1 ratio of reaction sites tointerrogation areas) the total number of reaction sites would be back upto 384 (i.e., equal to the number of wells in a conventional 384 wellSBS microtiter plate). Using microfluidics production technology, arraysof reaction sites can be constructed with 2, 3, 4, 6, 8, 10, 11, 12, 20,or more reaction sites overlapping each SBS-formatted interrogationarea. This results in throughputs that are double, triple, quadruple,etc., those of a conventional SBS-formatted microtiter plate.

For example, FIG. 2 shows a portion of a reaction site array 202 whereeach SBS-formatted interrogation area 208 encompasses four reactionsites 206 a-d. The 4:1 ratio of reaction sites to interrogation areasgives the reaction site array 202 twice the number of reaction sites(i.e., 768 reaction sites) versus a conventional 384 well SBS-formattedmicrotiter plate. Each reaction site 206 may be individually addressableby one or more fluid flow lines, including sample lines 212 and reagentlines 213. The lines act as channels to guide the flow of a fluid from areservoir to a reaction site. Flow through the fluid flow lines may alsobe controlled by control lines 214. These lines 214 can be pressureactuated to open and close the fluid flow lines to form a fluid pathfrom the reservoir to the reaction site. The lines 214 can also controlthe amount of fluid that enters the reaction site.

The 2×2 unit cells of reaction sites 206 a-d encompassed by theinterrogation areas 208 can boost the efficiency of SBS-formattedequipment a number of ways. For example, SBS-formatted screeningequipment can monitor sequential reactions that would be run at each ofthe reaction sites 206 a-d and then read. This may start with a sampleand reagent being introduced to the first reaction site 206 a andreading the results (e.g., a change in absorbance or fluorescence causedby a product species). The first site 206 a may be bleached or washedafter the run. Then, a second sample and reagent may be introduced tothe second reaction site 206 b while reading the results of thiscombination. The process may then be repeated for the third site 206 cand, finally, the fourth reaction site 206 d.

The plate has 192 interrogation areas that each have 4 reaction sites,allowing a total of 768 reactions to be run on the plate, eithersimultaneously or sequentially. This is twice the number of reactionspossible using a conventional 384 well microtiter plate. Still morereactions are possible by packing 5, 6, 7, 8, 9, 10, etc., reactionsites 206 into each of the interrogation areas 208.

Additional methods include running reactions in all four reaction sites206 a-d simultaneously instead of sequentially. For example, reagentsmay be labeled with different colored dyes in each of the reaction sites206 a-d that absorb and/or fluoresce light of different coloredwavelengths. Spectrally discriminating detection equipment that monitorseach wavelength independently can detect changes in all four reactionsites at the same time. These multiple color reactions can significantlyincrease the throughput rate of for an SBS-formatted reaction plate byrunning two, three, four, five, etc., times the number of reactions inthe same period of time as a conventional 384 well microtiter plate.

There are also methods and processes for using reaction sites 206 tomake reaction product samples that can be transported to product samplereservoirs in the reservoir arrays 110 on plate 100. SBS-formattedequipment (e.g., pipetting robots) can be programmed to automaticallyextract the product samples from the reservoirs and place them instorage containers or instrumentation for additional chemical and/orbiological analysis.

Embodiments of these methods may also include multi-stage reactions andsyntheses where reaction products from one reaction site are actuallyintermediates in a more complex reaction scheme. In these methods, afirst reaction product formed from reactants in a first reaction site206 are combined with additional reagents and/or reaction products froma second reaction site. The products made by reacting the first reactionproduct with the additional reactants and/or reaction products may bethe final reaction products that are transported to the product samplereservoir. The methods may include even more complexity by forming aplurality of reaction product intermediates in multiple reaction sites206 that are combined at various stages leading to a final product orgroup of products.

At each reaction site, SBS-formatted equipment may be used to monitorand/or influence the reactions taking place in the reaction sites 206.For example, spectral analysis equipment may be used to monitor theconsumption or production of chemical species in a reaction site 206.Equipment may also be used to apply localized heating and/or lightexposure (e.g., UV light exposure) to influence the types of reactionproducts that are generated.

Further methods include using the reaction sites 206 in high-throughputmolecular genetics analyses. For example, SBS-formatted plate 100 may beused with a standard real-time PCR instrument to perform an automatedDNA analysis of single nucleotide polymorphisms (SNPs) from one or moresamples of genetic material. Multiple SNP reactions (e.g., SNPrestriction fragment length polymorphism) may be performed in reactionsites 206 and the reaction products amplified by polymerase chainreaction (PCR). The PCR product samples may then be transported toproduct reservoirs 110 where automated DNA analysis equipment canextract the samples and run them through electrophoresis equipment(e.g., gel or capillary electrophoresis equipment).

Fluids may be supplied via supply channels (not shown) to the reactionsites 106 in the reaction site array 102 from the reservoir arrays 110a-b. The reservoir arrays 110 a-b may be filed with samples and reagentsthat are delivered in various combinations to the reaction sites 106. Asshown in FIG. 4, the fluids are delivered through fluid channels 412 inthe reaction site array 402. The flow of fluids through the fluidchannels 412 are controlled by pressure actuation of intersectingcontrol channels 414. Increasing the pressure in a control channel 414causes it to dilate and close the flow channel at a point where thechannels intersect. Conversely, decreasing the pressure in thepressurized control valve causes it to constrict and reopen the flowchannel. A series of control channels can be configured into aperistaltic pump (not shown) to transport fluids through the flowchannels.

By selectively actuating the control channels 414, specific combinationsof fluid samples from the fluid reservoir array (not shown) may be addedto individual reaction sites 406. For example, a first reservoir arraymay hold M samples and a second reservoir array may hold N reagents.Selective actuation of the control channels 414 may be done to delivercombinations of each M sample and N reagent to one of the N×M discretereaction sites 406. Measurements may then be taken to determine how eachcombination of sample and reagent react.

FIG. 5 shows an exploded view of a microfluidics plate 500. In thisview, the reaction site array 502 is shown separated from the rest ofplate 500. Embodiments of the invention are contemplated where the array502 may be detached from the rest of the plate 500, allowing multiplereaction site arrays 502 to be cycled through the device. The plate 500and/or array 502 may include an asymmetrical shape or alignment notch tohelp couple connectors for the flow and control lines in the array 502with the complementary connectors on plate 500 when the pieces arejoined. An array 502 that has been used in the plate 500 may bepermanently disposed of, or if possible, recycled using acceptedcleaning and/or sterilization procedures. In other embodiments thereaction site array 502 may be permanently affixed or formed integralwith plate 500.

Exemplary Device Optics

Optical lenses may be used to position and concentrate light on thereactions sites in the microfluidic plates. This can increase the signalsensitivity and/or reduce the cross-talk in spectroscopic detectionequipment measuring spectral events (e.g., absorption spectroscopy,fluorescence spectroscopy, etc.). The lenses can also concentrate lightthat may be used to enhance photochemical reactions in a selectedreaction site.

The lenses may be integral to the microfluidic plate, for example,present within the elastomeric structure of the device. An integratedlens may be formed from any material that has an index of refractionthat is different from the surrounding material. In certain embodiments,the lens is provided by molding a generally bi-convex-shaped void intothe structure of the elastomeric material. Air or another fluid ispumped into the void such that it inflates to provide a pre-determinedbi-convex shape within the elastomeric material that acts as a lens.Alternatively, the space may be evacuated to cause the surface layer torecess into the plate and form a concave lens. Adjustments in thepressure may be made to adjust the curvature and optical properties ofthe lenses, such as the focal point. The integral lenses may also beused with external lenses to form compound lenses. In certainembodiments, two or more integral lenses are provided to provide amulti-lens system within the device. Integral lenses are generallypositioned within the device above a reaction site so as to provideoptical magnification of the reaction site. In certain embodiments, theintegrated lens is inflated with a gas, such as air, and in additionalembodiments, the lens may be inflated using a liquid such as water,saline, an oil, an alcohol, a polyol, an isobutyrate, etc. The inflatingfluid may be selected to provide the desired optical (e.g., lightfocusing) properties.

As shown in FIG. 3A, a biconvex focusing lens 302 for focusing a lightsource into the area of an individual reaction site 306. Concentratingthe detection light on the reaction site can increase the strength of anabsorbance and/or fluorescence signal from the site. Also, focusing thelight source reduces the intensity of stray and scatted light that mayinterfere with measurements in nearby reactions sites (i.e.,cross-talk). FIG. 3B shows a biconcave lens 308 that may be used todiffuse the source light more evenly across a plurality of reactionsites 310 a-b. These lenses may provide a more uniform distribution ofsource light across multiple reaction sites 310 that are packed into aninterrogation area.

Additional types of lenses (not shown) may also be used to control theposition and concentration of light impinging on the microfluidicsplate. For example, plano-covex lenses, convex-concave lenses, meniscuslenses, plano-concave lenses, and fresnel lenses may also be used withthe invention. The lenses may be used alone (e.g., a simple lens) or incombination (e.g., a compound lens) to control the focus of the lightimpinging on, or emitted from, the microfluidics plate.

The SBS Dimensional Standards for Microplates

The Society for Biomolecular Screening (“SBS”) has developed formattingstandards for microplates used in high throughput screening processesfor biological and chemical compounds. These automated processesincluded the use of robot pipetting to transfer fluid samples to anarray of reaction wells formed in the microplate. Detection equipmentwas aligned with the wells to observe and measure events (e.g., chemicalreactions, enzymatic catalysis, crystallizations, etc.). As the numberof vendors and systems proliferated, standards were clearly needed toaddress compatibility problems. SBS developed dimensional standards formicroplates that are followed by a significant number of microplatemanufacturers and instrument makers that utilize microplates.

SBS has defined dimensional standards for 96, 384, and 1536 wellmicroplates. In each case, the microplate has a rectangular shape thatmeasures 127.76 mm±0.5 mm in length by 85.48 mm±0.5 mm in width. Thefour corners of the plate are rounded with a corner radius to theoutside of 3.18±1.6 mm. The complete definitions for these standardswere published by the American National Standards Institute on Mar. 28,2005, in publications ANSI/SBS 1-2004, ANSI/SBS 2-2004; ANSI/SBS 3-2004;and ANSI/SBS 4-2004, the entire contents of which are hereinincorporated by reference for all purposes. A summary of the definitionsfor 96, 384 and 1536 well plates are provided here:

The 96 Well Format

FIG. 6A shows an arrangement for a 96 well microplate, arranged in an 8row by 12 column rectangular array. The columns of the array are definedby the distance between the left outside edge of the plate and thecenter of the first column of wells being 14.38 mm. Each additionalcolumn is an additional 9 mm in distance from the left outside edge ofthe plate. The top edge of the part is defined as the two 12.7 mm areasmeasured from the corners of the plate. The rows of the 96 well arrayare defined by a distance of 11.24 mm between the top outside edge ofthe plate and the center of the first row of wells. Each additional rowis an additional 9 mm from the top outside edge of the plate. The topedge of the part is defined as the two 12.7 mm areas measured from thecorners of the plate.

The 384 Well Format

FIG. 6B shows an arrangement for a 384 well microplate, arranged in an16 row by 24 column rectangular array. The columns of the array aredefined by the distance between the left outside edge of the plate andthe center of the first column of wells being 12.13 mm. Each additionalcolumn is an additional 4.5 mm in distance from the left outside edge ofthe plate. The top edge of the part is defined as the two 12.7 mm areasmeasured from the corners of the plate. The rows of the 384 well arrayare defined by a distance of 8.99 mm between the top outside edge of theplate and the center of the first row of wells. Each additional row isan additional 4.5 mm from the top outside edge of the plate. The topedge of the part is defined as the two 12.7 mm areas measured from thecorners of the plate.

The 1536 Well Format

FIG. 6C shows an arrangement for a 1536 well microplate, arranged in an32 row by 48 column rectangular array. The columns of the array aredefined by the distance between the left outside edge of the plate andthe center of the first column of wells being 11.005 mm. Each additionalcolumn is an additional 2.25 mm in distance from the left outside edgeof the plate. The top edge of the part is defined as the two 12.7 mmareas measured from the corners of the plate. The rows of the 1536 wellarray are defined by a distance of 7.865 mm between the top outside edgeof the plate and the center of the first row of wells. Each additionalrow is an additional 2.25 mm from the top outside edge of the plate. Thetop edge of the part is defined as the two 12.7 mm areas measured fromthe corners of the plate.

Carry-on Mixing and Metering in the SBS Formatted Devices

This invention further relates to microfluidic devices and methods ofusing the devices that provide precise metering of fluid volumes andefficient mixing of the metered volumes. The devices are useful foranalytical assays for research or diagnostic purposes where highdensity, high throughput, sample parsimony, and lower cost are desired.The devices and methods are also useful as tools for the synthesis,sorting, and storage of high value chemical and biological entities. Theapplication of microfluidic devices with high sensitivity andreproducibility may require the carry-on mixing and metering approach ofthe present invention in order to achieve practical and enhancedsensitivity and dynamic range to be of practical value in a researchsetting.

The microfluidic devices of the present invention may also utilize aconfiguration in which at least one solution is metered into a segmentof a flow channel, typically through a junction disposed between valvesalong the flow channel. The junction typically has an on/off valve or aone way check valve at the inlet portion of the junction. The flowchannel valves that bracket the junction are closed and the junctioninlet valve is opened. A solution is instilled into the segment of theflow channel. The filling of the segment is preferably performed by“blind filling” the segment. Blind filling takes advantage of thepermeability of the material defining at least one side of the flowchannel to gases and not to liquid. The first solution is filled intothe flow channel segment by placing the solution under pressure at thejunction and allowing the first solution to fill the segment as thegases that are present in the flow channel diffuses out through the gaspermeable material. Once the segment of the flow channel defined by thevalves is filled, the inlet junction is closed or allowed to close and aprecisely defined volume is contained within the flow channel segment.The exact volume is determined by the flow channel dimensions and thespacing of the valves along the segment that are closed to define theblind filled portion of the flow channel. With the valves remainingclosed, a second solution is introduced into an empty portion of theflow channel by blind filling against one of the closed valves. Bymaintaining the second solution under pressure and then opening thevalves on the flow channel segment, the second solution pushes the firstsolution through the flow channel. In a preferred embodiment, the flowchannel segment valve opposite the valve against which the secondsolution is blind filled, is adjacent to a reaction well of a definedvolume. When the flow channel segment valves are opened, the secondsolution pushes the first solution into the reaction well. Byfabricating the reaction well such that the reaction well volume isgreater that the volume of the first solution in the flow channel, aprecisely defined amount of the second solution is pushed into thereaction well along with the known volume of the first solution. Thevolume of the second solution that fills the reaction well is defined bythe volume of the reaction well minus the volume of the first solution.As both solutions fill the reaction well, mixing of the solutionsoccurs. The mixing of the solutions is typically efficient and isgreater that 25% efficient, preferably greater than 35% efficient, morepreferably greater than 50% efficient, more preferably greater that 65%efficient, more preferably greater than 75% efficient, more preferablygreater than 85% efficient, more preferably greater than 90% efficient,more preferably greater than 95% efficient, more preferably greater that99% efficient, and more preferably about 100% efficient. In oneembodiment, the reaction well has only the flow channel entering thechamber defined by its volume. In another embodiment, the reaction wellhas a separate opening for an additional inlet or outlet channel. In anadditional embodiment, the reaction well has a plurality of openings forother channels in addition to the described flow channel.

Various terms such as “pulse-chase,” “carry-on,” slug mixing,” pigmixing,” and “bolus mixing” may be used to describe the described mixingtechnique.

Microfluidic Chip Construction Preferred Layer, Channel, and ReactionWell Dimensions:

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels preferably have width-to-depthratios of about 10:1. A non-exclusive list of other ranges ofwidth-to-depth ratios in accordance with embodiments of the presentinvention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels have widths of about 1 to 1000 microns. Anon-exclusive list of other ranges of widths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels have depths of about 1 to 100 microns. A non-exclusivelist of other ranges of depths of flow channels in accordance withembodiments of the present invention is 0.01 to 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250 microns, andmore preferably 1 to 100 microns, more preferably 2 to 20 microns, andmost preferably 5 to 10 microns. Exemplary channel depths includeincluding 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm,3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, and 250μm.

Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, a layer is 50 microns to over a centimeter thick,and more preferably approximately 4 mm thick. A non-exclusive list ofranges of thickness of the elastomer layer in accordance with otherembodiments of the present invention is between about 0.1 micron to 1cm, 1 micron to 1 cm, 10 microns to 0.5 cm, 100 microns to 10 mm.

Accordingly, membranes separating flow channels have a typical thicknessof between about 0.01 and 1000 microns, more preferably 0.05 to 500microns, more preferably 0.2 to 250, more preferably 1 to 100 microns,more preferably 2 to 50 microns, and most preferably 5 to 40 microns.Exemplary membrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm,75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm,and 1000 μm.

Furthermore, embodiments of the present invention provide reducedreaction volumes. In embodiments of the present invention, reactionvolumes ranging from 10 picoliters to 100 nanoliters are utilized. Insome embodiments, reaction volumes greater than 100 nanoliters areutilized. Reaction wells may also be in the microliter, nanoliter,picoliter, femtoliter or lower range of volume. In one embodiment, thereaction well volume is between 1-1000 femtoliters. Merely by way ofexample, in an embodiment, the methods and systems of the presentinvention are utilized with reaction volumes of 10 picoliters, 50picoliters, 100 picoliters, 250 picoliters, 500 picoliters, and 1nanoliter. In alternative embodiments, reaction volumes of 2 nanoliters,5 nanoliters, 10 nanoliters, 20 nanoliters, 30 nanoliters, 40nanoliters, 50 nanoliters, 75 nanoliters, and 100 nanoliters areutilized. In another embodiment, the reaction well volume is between1-1000 picoliters. In another embodiment, the reaction well volume isbetween 0.01-100 nanoliters, preferably between 1-75 nanoliters. In oneembodiment the reaction well volume is about 50 nanoliters. In oneembodiment the reaction well volume is about 7.6 nanoliters. In anotherembodiment, the reaction well volume is 6 nL. The volume defined for thefirst solution in the flow channel (the slug volume or carry-on volume)is a fraction of the reaction well volume. In various embodiments, thefraction may be ⅞, ¾, ⅝, ½, ⅜, ¼, ⅕, ⅛, 1/10, 1/12, 1/20, 1/25, 1/50,1/100, or less of the total reaction well volume.

Multilayer Soft Lithography Construction Techniques and Materials: SoftLithographic Bonding:

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In one aspect, the various layers of elastomer are bound together in aheterogenous bonding in which the layers have a different chemistry.Alternatively, a homogenous bonding may be used in which all layerswould be of the same chemistry. Thirdly, the respective elastomer layersmay optionally be glued together by an adhesive instead. In a fourthaspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Elastomeric layers may be created by spin-coating an RTV mixture onmicrofabricated mold at 2000 rpm for 30 seconds yielding a thickness ofapproximately 40 microns. Additional elastomeric layers may be createdby spin-coating an RTV mixture on microfabricated mold. Both layers maybe separately baked or cured at about 80° C. for 1.5 hours. Theadditional elastomeric layer may be bonded onto first elastomeric layerat about 80° C. for about 1.5 hours.

Suitable Elastomeric Materials:

Allcock et al, Contemporary Polymer Chemistry, 2nd Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, elastomeric layers maypreferably be fabricated from silicone rubber. However, other suitableelastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, polybutadiene, polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (≈1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thepolyisobutylene backbone, which may then be vulcanized as above.

Poly(Styrene-Butadiene-Styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

Cross Linking Agents:

In addition to the use of the simple “pure” polymers discussed above,crosslinking agents may be added. Some agents (like the monomers bearingpendant double bonds for vulcanization) are suitable for allowinghomogeneous (A to A) multilayer soft lithography or photoresistencapsulation; in such an approach the same agent is incorporated intoboth elastomer layers. Complementary agents (i.e. one monomer bearing apendant double bond, and another bearing a pendant Si—H group) aresuitable for heterogeneous (A to B) multilayer soft lithography. In thisapproach complementary agents are added to adjacent layers.

Other Materials:

In addition, polymers incorporating materials such as chlorosilanes ormethyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) suchas Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or In 245 from UCBChemical may also be used.

The following is a non-exclusive list of elastomeric materials which maybe utilized in connection with the present invention: polyisoprene,polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and siliconepolymers; or poly(bis(fluoroaLkoxy)phosphazene) (PNF, Eypel-F),poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(1-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon).

Doping and Dilution:

Elastomers may also be “doped” with uncrosslinkable polymer chains ofthe same class. For instance RTV 615 may be diluted with GE SF96-50Silicone Fluid. This serves to reduce the viscosity of the uncuredelastomer and reduces the Young's modulus of the cured elastomer.Essentially, the crosslink-capable polymer chains are spread furtherapart by the addition of “inert” polymer chains, so this is called“dilution”. RTV 615 cures at up to 90% dilution, with a dramaticreduction in Young's modulus.

Other examples of doping of elastomer material may include theintroduction of electrically conducting or magnetic species, asdescribed in detail below in conjunction with alternative methods ofactuating the membrane of the device. Should it be desired, doping withfine particles of material having an index of refraction different thanthe elastomeric material (i.e. silica, diamond, sapphire) is alsocontemplated as a system for altering the refractive index of thematerial. Strongly absorbing or opaque particles may be added to renderthe elastomer colored or opaque to incident radiation. This mayconceivably be beneficial in an optically addressable system.

Finally, by doping the elastomer with specific chemical species, thesedoped chemical species may be presented at the elastomer surface, thusserving as anchors or starting points for further chemicalderivitization.

In the present application, references are made to certain types of“reaction” chambers or wells in a microfluidic device. In general and inaddition to those characteristics described above, these includeprocessing sites, processing chambers, and/or reaction sites, anycombination of these, and the like. These chambers may be closed,partially closed, open, partially open, sealed, or combinations thereof,including any temporary or transient conditions involving any of thesestates, and the like. In some embodiments, the chambers are sealed,capable of being sealed, closeable, isolated, capable of being isolated,and combinations thereof, and any combination or single condition of anytemporary or transient conditions involving any of these states, and thelike. Therefore, use of the term reaction chamber is not intended tolimit the present invention, but to include these other structures.Additionally, the term chamber is not intended to limit the presentinvention, but should be used in its ordinary meaning, unless specificfeatures associated with the chamber have been recited. Of course, therecan be other variations, modifications, and alternatives.

The microfluidic devices that are described herein are furthercharacterized in part in some embodiments by utilizing variouscomponents such as flow channels, control channels, valves and/or pumpsfabricated from elastomeric materials. In some instances, essentiallythe entire device is made of elastomeric materials. Consequently, suchdevices differ significantly in form and function from the majority ofconventional microfluidic devices that are formed from plastics orsilicon-based materials. The number of reaction chambers provided variesaccording to embodiments of the present invention. Other microfluidicdevices fabricated by non elastomeric materials such as glass, silicon,rigid plastics, and metal are possible, however, elastomericmicrofluidic devices are a preferred embodiment of the invention.

The design of the devices enables them to be utilized in combinationwith a number of different heating systems. Thus, the devices are usefulin conducting diverse analyses that require temperature control.Additionally, those microfluidic devices adapted for use in heatingapplications can incorporate a further design feature to minimizeevaporation of sample from the reaction sites. Devices of this type ingeneral include a number of guard channels and/or reservoirs or chambersformed within the elastomeric device through which water can be flowedto increase the water vapor pressure within the elastomeric materialfrom which the device is formed, thereby reducing evaporation of samplematerial from the reaction sites.

In another embodiment, a temperature cycling device may be used tocontrol the temperature of the microfluidic devices. Preferably, themicrofluidic device would be adapted to make thermal contact with themicrofluidic device. Where the microfluidic device is supported by asubstrate material, such as a glass slide or the bottom of a carrierplate, such as a plastic carrier, a window may be formed in a region ofthe carrier or slide such that the microfluidic device, preferably adevice having an elastomeric block, may directly contact theheating/cooling block of the temperature cycling device. In a preferredembodiment, the heating/cooling block has grooves therein incommunication with a vacuum source for applying a suction force to themicrofluidic device, preferably a portion adjacent to where thereactions are taking place. Alternatively, a rigid thermally conductiveplate may be bonded to the microfluidic device that then mates with theheating and cooling block for efficient thermal conduction resulting.

The array format of certain of the devices means the devices can achievehigh throughput. Collectively, the high throughput and temperaturecontrol capabilities make the devices useful for performing largenumbers of nucleic acid amplifications (e.g., polymerase chain reaction(PCR)). Such reactions will be discussed at length herein asillustrative of the utility of the devices, especially of their use inany reaction requiring temperature control. However, it should beunderstood that the devices are not limited to these particularapplications. The devices can be utilized in a wide variety of othertypes of analyses or reactions. Examples include analyses ofprotein-ligand interactions and interactions between cells and variouscompounds. Further examples are provided throughout the presentspecification.

The microfluidic devices disclosed herein are typically constructed atleast in part from elastomeric materials and constructed by single andmultilayer soft lithography (MSL) techniques and/or sacrificial-layerencapsulation methods (see, e.g., Unger et al. (2000) Science288:113-116, and PCT Publication WO 01/01025, both of which areincorporated by reference herein in their entirety for all purposes).Utilizing such methods, microfluidic devices can be designed in whichsolution flow through flow channels of the device is controlled, atleast in part, with one or more control channels that are separated fromthe flow channel by an elastomeric membrane or segment. This membrane orsegment can be deflected into or retracted from the flow channel withwhich a control channel is associated by applying an actuation force tothe control channels. By controlling the degree to which the membrane isdeflected into or retracted out from the flow channel, solution flow canbe slowed or entirely blocked through the flow channel. Usingcombinations of control and flow channels of this type, one can preparea variety of different types of valves and pumps for regulating solutionflow as described in extensive detail in Unger et al. (2000) Science288:113-116, and PCT Publication WO/02/43615 and WO 01/01025.

If the device is to be utilized in temperature control reactions (e.g.,thermocycling reactions), then, as described in greater detail infra,the elastomeric device is typically fixed to a support (e.g., a glassslide). The resulting structure can then be placed on a temperaturecontrol plate, for example, to control the temperature at the variousreaction sites. In the case of thermocycling reactions, the device canbe placed on any of a number of thermocycling plates.

Because the devices are made of elastomeric materials that arerelatively optically transparent, reactions can be readily monitoredusing a variety of different detection systems at essentially anylocation on the microfluidic device. Most typically, however, detectionoccurs at the reaction site itself (e.g., within a region that includesan intersection of flow channels or at the blind end of a flow channel).The fact that the device is manufactured from substantially transparentmaterials also means that certain detection systems can be utilized withthe current devices that are not usable with traditional silicon-basedmicrofluidic devices. Detection can be achieved using detectors that areincorporated into the device or that are separate from the device butaligned with the region of the device to be detected.

Some high-density matrix designs utilize fluid communication viasbetween layers of the microfluidic device to weave control lines andfluid lines through the device. For example, by having a fluid line ineach layer of a two layer elastomeric block, higher density reactioncell arrangements are possible. As will be evident to one of skill inthe art, multilayer devices allow fluid lines to cross over or undereach other without being in fluid communication. The microfluidicdevices utilized in embodiments of the present invention may be furtherintegrated into the carrier devices described in co-pending and commonlyowned U.S. patent application Ser. No. 11/058,106 by Unger filed on Feb.14, 2005, which is incorporated herein for all purposes. The carrier ofUnger provides on-board continuous fluid pressure to maintain valveclosure away from a source of fluid pressure, e.g., house air pressure.Unger further provides for an automated system for charging andactuating the valves of the present invention as described therein. Ananother preferred embodiment, the automated system for chargingaccumulators and actuating valves employs a device having a platen thatmates against one or more surfaces of the microfluidic device, whereinthe platen has at least two or more ports in fluid communication with acontrolled vacuum or pressure source, and may include mechanicalportions for manipulating portions of the micro fluidic device, forexample, but not limited to, check valves. Check valves may beincorporated into the chip design to provide for a one-way flow of fluid(either reagent, sample, first reactant, second reactant, thirdreactant, etc.). Check valves and their fabrication are described incommonly owned U.S. Provisional Application Ser. No. 60/849,223 by Wangfiled on Oct. 4, 2006 which is incorporated herein for all purposes.

Another device utilized in embodiments of the present invention providesa carrier used as a substrate for stabilizing an elastomeric block.Preferably the carrier has one or more of the following features; a wellor reservoir in fluid communication with the elastomeric block throughat least one channel formed in or with the carrier; an accumulator influid communication with the elastomeric block through at least onechannel formed in or with the carrier; and, a fluid port in fluidcommunication with the elastomeric block, wherein the fluid port ispreferably accessible to an automated source of vacuum or pressure, suchas the automated system described above, wherein the automated sourcefurther comprises a platen having a port that mates with the fluid portto form an isolated fluid connection between the automated system forapplying fluid pressure or vacuum to the elastomeric block. In devicesutilized in certain embodiments, the automated source can also makefluid communication with one or more accumulators associated with thecarrier for charging and discharging pressure maintained in anaccumulator. In certain embodiments, the carrier may further comprise aregion located in an area of the carrier that contacts the microfluidicdevice, wherein the region is made from a material different fromanother portion of the carrier, the material of the region beingselected for improved thermal conduction and distribution propertiesthat are different from the other portion of the carrier. Preferredmaterials for improved thermal conduction and distribution include, butare not limited to silicon, preferably silicon that is highly polished,such as the type of silicon available in the semiconductor field as apolished wafer or a portion cut from the wafer, e.g., chip.

As described more fully below, embodiments of the present inventionutilize a thermal source, for example, but not limited to a PCRthermocycler, which may have been modified from its originalmanufactured state. Generally the thermal source has a thermallyregulated portion that can mate with a portion of the carrier,preferably the thermal conduction and distribution portion of thecarrier, for providing thermal control to the elastomeric block throughthe thermal conduction and distribution portion of the carrier. In apreferred embodiment, thermal contact is improved by applying a sourceof vacuum to a one or more channels formed within the thermallyregulated portion of the thermal source, wherein the channels are formedto contact a surface of the thermal conduction and distribution portionof the carrier to apply suction to and maintain the position of thethermal conduction and distribution portion of the carrier. In apreferred embodiment, the thermal conduction and distribution portion ofthe carrier is not in physical contact with the remainder of thecarrier, but is associated with the remainder of the carrier and theelastomeric block by affixing the thermal conduction and distributionportion to the elastomeric block only and leaving a gap surrounding theedges of the thermal conduction and distribution portion to reduceparasitic thermal effects caused by the carrier. It should be understoodthat in many aspects of the invention described herein, the preferredelastomeric block could be replaced with any of the known microfluidicdevices in the art not described herein, for example devices producedsuch as the GeneChip® by Affymetrix® of Santa Clara, Calif., USA, or byCaliper of Mountain View, Calif., USA. U.S. patents issued to Soane,Parce, Fodor, Wilding, Ekstrom, Quake, or Unger, describe microfluidicor mesoscale fluidic devices that can be substituted for the elastomericblock of the present invention to take advantage of the thermaladvantages and improvements, e.g., suction positioning, reducingparasitic thermal transfer to other regions of the fluidic device, whichare described above in the context of using an elastomeric block.

Utilizing systems and methods provided according to embodiments of thepresent invention, throughput increases are provided over 384 wellsystems. As an example, throughput increases of a factor of 4, 6, 12,and 24 and greater are provided in some embodiments. These throughputincreases are provided while reducing the logistical friction ofoperations. Moreover the systems and methods of embodiments of thepresent invention enable multiple assays for multiple samples. Forexample, in a specific embodiment 24 samples and 24 assays are utilizedto provide a total of 576 data points. In another embodiment, 32 samplesand 32 assays are utilized to provide a total of 1024 data points. Inanother embodiment, 48 samples and 48 assays are utilized to provide2304 data points. In another embodiment, 96 samples and 48 assays areutilized to provide 4608 data points. In another embodiment, 96 samplesand 96 assays are utilized to provide a total of 9,216 data points. In aparticular example, the 96 assays are components of a TaqMan 5′ NucleaseAssay.

Depending on the geometry of the particular microfluidic device and thesize of the microfluidic device and the arrangement of the fluidcommunication paths and processing site, embodiments of the presentinvention provide for a range of processing site (or reaction chamber)densities. In some embodiments, the methods and systems of the presentinvention are utilized with chamber densities ranging from about 100chambers per cm² to about 1 million chambers per cm². Merely by way ofexample, microfluidic devices with chamber densities of 250, 1,000,2,500, 10,000, 25,000, 100,000, and 250,000 chambers per cm² areutilized according to embodiments of the present invention. In someembodiments, chamber densities in excess of 1,000,000 chambers per cm²are utilized, although this is not required by the present invention.

Operating microfluidic devices with such small reaction volumes reducesreagent usage as well as sample usage. Moreover, some embodiments of thepresent invention provide methods and systems adapted to performreal-time detection, when used in combination with real-timequantitative PCR. Utilizing these systems and methods, three, four,five, or six orders of linear dynamic range are provided for someapplications as well as quantitative resolution high enough to allow forthe detection of sub-nanoMolar fluorophore concentrations in 10nanoliter volumes.

Smart Chip Assays and Reactions

Through the present application, references are made to fluorescentindications from a microfluidic device. Included within the scope of thepresent invention are not only fluorescent indications, but luminescentindications, including chemiluminescent, electroluminescent,electrochemiluminescent, and phospholuminescent, bioluminescent, andother luminescent processes, or any other processing involving any othertype of indications that may be detected using a detection device. Aswill be evident to one of skill in the art, methods and systems operablein the detection and analysis of these fluorescent and luminescentindications are transferable from one indication to another.Additionally, although some embodiments of the present invention utilizespectral filters as optical elements, this is not required by thepresent invention. Some fluorescent and luminescent applications do notutilize spectral filters in the optical excitation path, the opticalemission path, or both. As described herein, other embodiments utilizespectral filters. One of skill in the art will appreciate thedifferences associated with particular applications.

In some embodiments, a variety of devices and methods for conductingmicrofluidic analyses are utilized herein, including devices that can beutilized to conduct thermal cycling reactions such as nucleic acidamplification reactions. The devices differ from conventionalmicrofluidic devices in that they include elastomeric components; insome instances, much or all of the device is composed of elastomericmaterial. For example, amplification reactions can be linearamplifications, (amplifications with a single primer), as well asexponential amplifications (i.e., amplifications conducted with aforward and reverse primer set).

The methods and systems provided by some embodiments of the presentinvention utilize blind channel type devices in performing nucleic acidamplification reactions. In these devices, the reagents that aretypically deposited within the reaction sites are those reagentsnecessary to perform the desired type of amplification reaction. Usuallythis means that some or all of the following are deposited: primers,polymerase, nucleotides, metal ions, buffer, and cofactors, for example.The sample introduced into the reaction site in such cases is thenucleic acid template. Alternatively, however, the template can bedeposited and the amplification reagents flowed into the reaction sites.As discussed in more detail throughout the present specification, when amatrix device is utilized to conduct an amplification reaction, samplescontaining nucleic acid template are flowed through the vertical flowchannels and the amplification reagents through the horizontal flowchannels or vice versa.

PCR is perhaps the best known amplification technique. The devicesutilized in embodiments of the present invention are not limited toconducting PCR amplifications. Other types of amplification reactionsthat can be conducted include, but are not limited to, (i) ligase chainreaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989) and Landegrenet al., Science 241:1077 (1988)); (ii) transcription amplification (seeKwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); (iii)self-sustained sequence replication (see Guatelli et al., Proc. Nat.Acad. Sci. USA, 87:1874 (1990)); and (iv) nucleic acid based sequenceamplification (NASBA) (see, Sooknanan, R. and Malek, L., BioTechnology13: 563-65 (1995)). Each of the foregoing references is incorporatedherein by reference in their entirety for all purposes.

Moreover, certain devices are designed to conduct thermal cyclingreactions (e.g., PCR) with devices that include one or more elastomericvalves to regulate solution flow through the device. Thus, methods forconducting amplification reactions with devices of this design are alsoprovided.

Amplification products (amplicons) can be detected and distinguished(whether isolated in a reaction chamber or at any subsequent time) usingroutine methods for detecting nucleic acids. Many different signalmoieties may be used in various embodiments of the present invention.For example, signal moieties include, but are not limited to,fluorophores, radioisotopes, chromogens, enzymes, antigens, heavymetals, dyes, phosphorescence groups, chemiluminescent groups, andelectrochemical detection moieties. Exemplary fluorophores that may beused as signal moieties include, but are not limited to, rhodamine,cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, VIC™, LIZ™, Tamra™,5-FAM™, 6-FAM™, and Texas Red (Molecular Probes). (VIC™, LIZ™, Tamra™,5-FAM™, and 6-FAM™ (all available from Applied Biosystems, Foster City,Calif.) Exemplary radioisotopes include, but are not limited to, ³²P,³³P, and ³⁵S. Signal moieties also include elements of multi-elementindirect reporter systems, e.g., biotin/avidin, antibody/antigen,ligand/receptor, enzyme/substrate, and the like, in which the elementinteracts with other elements of the system in order to effect adetectable signal. Certain exemplary multi-element systems include abiotin reporter group attached to a probe and an avidin conjugated witha fluorescent label. Detailed protocols for methods of attaching signalmoieties to oligonucleotides can be found in, among other places, G. T.Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif.(1996) and S. L. Beaucage et al., Current Protocols in Nucleic AcidChemistry, John Wiley & Sons, New York, N.Y. (2000).

Amplicons comprising double-stranded DNA can be detected usingintercalation dyes such as SYBR™, Pico Green (Molecular Probes, Inc.,Eugene, Oreg.), ethidium bromide and the like (see Zhu et al., 1994,Anal. Chem. 66:1941-48) and/or gel electrophoresis. More often,sequence-specific detection methods are used (i.e., amplicons aredetected based on their nucleotide sequence). Examples of detectionmethods include hybridization to arrays of immobilized oligo orpolynucleotides, and use of differentially labeled molecular beacons orother “fluorescence resonance energy transfer” (FRET)-based detectionsystems. FRET-based detection is a preferred method for detectionaccording to some embodiments of the present invention. In FRET-basedassays a change in fluorescence from a donor (reporter) and/or acceptor(quencher) fluorophore in a donor/acceptor fluorophore pair is detected.The donor and acceptor fluorophore pair are selected such that theemission spectrum of the donor overlaps the excitation spectrum of theacceptor. Thus, when the pair of fluorophores are brought withinsufficiently close proximity to one another, energy transfer from thedonor to the acceptor can occur and can be detected. A variety of assaysare known including, for example and not limitation, template extensionreactions, quantitative RT-PCR, Molecular Beacons, and Invader assays,these are described briefly below.

FRET and template extension reactions utilize a primer labeled with onemember of a donor/acceptor pair and a nucleotide labeled with the othermember of the donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during an template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods areparticularly useful in conducting single base pair extension reactionsin the detection of single nucleotide polymorphisms and are described inU.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719. The reactionscan optionally be thermocycled to increase signal using the temperaturecontrol methods and apparatus described throughout the presentspecification.

A variety of so-called “real time amplification” methods or “real timequantitative PCR” methods can also be used to determine the quantity ofa target nucleic acid present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantitation method which can be used successfully with the devicesdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan”method. See, for example, U.S. Pat. No. 5,723,591.

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye. In solution, the two end sectionscan hybridize with each other to form a hairpin loop. In thisconformation, the reporter and quencher dye are in sufficiently closeproximity that fluorescence from the reporter dye is effectivelyquenched by the quencher dye. Hybridized probe, in contrast, results ina linearized conformation in which the extent of quenching is decreased.Thus, by monitoring emission changes for the two dyes, it is possible toindirectly monitor the formation of amplification product. Probes ofthis type and methods of their use are described further, for example,by Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer,1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat.Biotechnol. 16:49-53 (1998).

The Scorpion detection method is described, for example, by Thelwell etal. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al., 2001,“Duplex Scorpion primers in SNP analysis and FRET applications” NucleicAcids Research 29:20. Scorpion primers are fluorogenic PCR primers witha probe element attached at the 5′-end via a PCR stopper. They are usedin real-time amplicon-specific detection of PCR products in homogeneoussolution. Two different formats are possible, the ‘stem-loop’ format andthe ‘duplex’ format. In both cases the probing mechanism isintramolecular. The basic elements of Scorpions in all formats are: (i)a PCR primer; (ii) a PCR stopper to prevent PCR read-through of theprobe element; (iii) a specific probe sequence; and (iv) a fluorescencedetection system containing at least one fluorophore and quencher. AfterPCR extension of the Scorpion primer, the resultant amplicon contains asequence that is complementary to the probe, which is renderedsingle-stranded during the denaturation stage of each PCR cycle. Oncooling, the probe is free to bind to this complementary sequence,producing an increase in fluorescence, as the quencher is no longer inthe vicinity of the fluorophore. The PCR stopper prevents undesirableread-through of the probe by Taq DNA polymerase.

Invader assays (Third Wave Technologies, Madison, Wis.) are usedparticularly for SNP genotyping and utilize an oligonucleotide,designated the signal probe that is complementary to the target nucleicacid (DNA or RNA) or polymorphism site. A second oligonucleotide,designated the Invader Oligo, contains the same 5′ nucleotide sequence,but the 3′ nucleotide sequence contains a nucleotide polymorphism. TheInvader Oligo interferes with the binding of the signal probe to thetarget nucleic acid such that the 5′ end of the signal probe forms a“flap” at the nucleotide containing the polymorphism. This complex isrecognized by a structure specific endonuclease, called the Cleavaseenzyme. Cleavase cleaves the 5′ flap of the nucleotides. The releasedflap binds with a third probe bearing FRET labels, thereby forminganother duplex structure recognized by the Cleavase enzyme. This timethe Cleavase enzyme cleaves a fluorophore away from a quencher andproduces a fluorescent signal. For SNP genotyping, the signal probe willbe designed to hybridize with either the reference (wild type) allele orthe variant (mutant) allele. Unlike PCR, there is a linear amplificationof signal with no amplification of the nucleic acid. Further detailssufficient to guide one of ordinary skill in the art are provided by,for example, Neri, B. P., et al., Advances in Nucleic Acid and ProteinAnalysis 3826:117-125, 2000) and U.S. Pat. No. 6,706,471.

A variety of multiplex amplification systems can be used in conjunctionwith the present invention. In one type, several different targets canbe detected simultaneously by using multiple differently labeled probeseach of which is designed to hybridize only to a particular target.Since each probe has a different label, binding to each target to bedetected based on the fluorescence signals. By judicious choice of thedifferent labels that are utilized, analyses can be conducted in whichthe different labels are excited and/or detected at differentwavelengths in a single reaction. See, e.g., Fluorescence Spectroscopy(Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al.,Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York,(1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules,2nd ed., Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Many diseases linked to genome modifications, either of the hostorganism or of infectious organisms, are the consequence of a change ina small number of nucleotides, frequently involving a change in a singlenucleotide. Such single nucleotide changes are referred to as singlenucleotide polymorphisms or simply SNPs, and the site at which the SNPoccurs is typically referred to as a polymorphic site. The devicesdescribed herein can be utilized to determine the identify of anucleotide present at such polymorphic sites. As an extension of thiscapability, the devices can be utilized in genotyping analyses.Genotyping involves the determination of whether a diploid organism(i.e., an organism with two copies of each gene) contains two copies ofa reference allele (a reference-type homozygote), one copy each of thereference and a variant allele (i.e., a heterozygote), or contains twocopies of the variant allele (i.e., a variant-type homozygote). Whenconducting a genotyping analysis, the methods of the invention can beutilized to interrogate a single variant site. However, as describedfurther below in the section on multiplexing, the methods can also beused to determine the genotype of an individual in many different DNAloci, either on the same gene, different genes or combinations thereof.

Genotyping analyses can be conducted using a variety of differentapproaches. In these methods, it is generally sufficient to obtain a“yes” or “no” result, i.e., detection need only be able to answer thequestion whether a given allele is present. Thus, analyses can beconducted only with the primers or nucleotides necessary to detect thepresence of one allele potentially at a polymorphic site. However, moretypically, primers and nucleotides to detect the presence of each allelepotentially at the polymorphic site are included.

Single Base Pair Extension (SBPE) reactions are one techniquespecifically developed for conducting genotyping analyses. Although anumber of SPBE assays have been developed, the general approach is quitesimilar. Typically, these assays involve hybridizing a primer that iscomplementary to a target nucleic acid such that the 3′ end of theprimer is immediately 5′ of the variant site or is adjacent thereto.Extension is conducted in the presence of one or more labelednon-extendible nucleotides that are complementary to the nucleotide(s)that occupy the variant site and a polymerase. The non-extendiblenucleotide is a nucleotide analog that prevents further extension by thepolymerase once incorporated into the primer. If the addednon-extendible nucleotide(s) is(are) complementary to the nucleotide atthe variant site, then a labeled non-extendible nucleotide isincorporated onto the 3′ end of the primer to generate a labeledextension product. Hence, extended primers provide an indication ofwhich nucleotide is present at the variant site of a target nucleicacid. Such methods and related methods are discussed, for example, inU.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and5,710,028; and in WO 92/16657.

Genotyping analyses can also be conducted using quantitative PCRmethods. In this case, differentially labeled probes complementary toeach of the allelic forms are included as reagents, together withprimers, nucleotides and polymerase. However, reactions can be conductedwith only a single probe, although this can create ambiguity as towhether lack of signal is due to absence of a particular allele orsimply a failed reaction. For the typical biallelic case in which twoalleles are possible for a polymorphic site, two differentially labeledprobes, each perfectly complementary to one of the alleles are usuallyincluded in the reagent mixture, together with amplification primers,nucleotides and polymerase. Sample containing the target DNA isintroduced into the reaction site. If the allele to which a probe iscomplementary is present in the target DNA, then amplification occurs,thereby resulting in a detectable signal as described in the detectionabove. Based upon which of the differential signal is, obtained, theidentity of the nucleotide at the polymorphic site can be determined. Ifboth signals are detected, then both alleles are present. Thermocyclingduring the reaction is performed as described in the temperature controlsection supra.

Gene expression analysis involves determining the level at which one ormore genes is expressed in a particular cell. The determination can bequalitative, but generally is quantitative. In a differential geneexpression analysis, the levels of the gene(s) in one cell (e.g., a testcell) are compared to the expression levels of the same genes in anothercell (control cell). A wide variety of such comparisons can be made.Examples include, but are not limited to, a comparison between healthyand diseased cells, between cells from an individual treated with onedrug and cells from another untreated individual, between cells exposedto a particular toxicant and cells not exposed, and so on. Genes whoseexpression levels vary between the test and control cells can serve asmarkers and/or targets for therapy. For example, if a certain group ofgenes is found to be up-regulated in diseased cells rather than healthycells, such genes can serve as markers of the disease and canpotentially be utilized as the basis for diagnostic tests. These genescould also be targets. A strategy for treating the disease might includeprocedures that result in a reduction of expression of the up-regulatedgenes.

The devices of the present application are not limited to biologicalassays such as PCR reactions. The efficient mixing of the carry-slugdesign is an enhancement to any chemical or biochemical assay and can beutilized in chemical synthesis (in particular where it is desirable tomonitor reaction progress or degree of completion of the reaction in anSBS formatted reader). Other assays that benefit from the presentinvention include immunological and enzymatic assays.

Exemplary Chip Holder

FIG. 7 shows an exploded view of a chip holder 11000 in accordance withone embodiment of the present invention. Bottom portion 11002 of chipholder 11000 includes raised peripheral portion 11004 surroundingrecessed area 11006 corresponding in size to the dimensions of chip11008, allowing microfluidic chip 11008 to be positioned therein.Peripheral region 11004 further defines screw holes 11010.

Microfluidic device 11008 is positioned within recessed area 11006 ofbottom portion 11002 of chip holder 11000. Microfluidic device 11008comprises an active region 11011 that is in fluidic communication withperipheral wells 11012 configured in first and second rows 11012 a and11012 b, respectively. Wells 11012 hold sufficient volumes of materialto allow device 11008 to function. Wells 11012 may contain, for example,solutions of crystallizing agents, solutions of target materials, orother chemical reagents such as stains. Bottom portion 11002 contains awindow 11003 that enables active region 11011 of chip 11008 to beobserved.

Top portion 11014 of chip holder 11000 fits over bottom chip holderportion 11002 and microfluidic chip 11008 positioned therein. For easeof illustration, in FIG. 80 top chip holder portion 11014 is showninverted relative to its actual position in the assembly. Top chipholder portion 11014 includes screw holes 11010 aligned with screw holes11010 of lower holder portion 11002, such that screws 11016 may beinserted through holes 11010 secure chip between portions 11002 and11014 of holder 11000. Chip holder upper portion 11014 contains a window11005 that enables active region 11011 of chip 11008 to be observed.

Lower surface 11014 a of top holder portion 11014 includes raisedannular rings 11020 and 11022 surrounding recesses 11024 and 11026,respectively. When top portion 11014 of chip holder 11000 is pressedinto contact with chip 11008 utilizing screws 11016, rings 11020 and11022 press into the soft elastomeric material on the upper surface ofchip 11008, such that recess 11024 defines a first chamber over top row11012 a of wells 11012, and recess 11026 defines a second chamber overbottom row 11012 b of wells 11012. Holes 11030 and 11032 in the side oftop holder portion 11014 are in communication with recesses 11024 and11026 respectively, to enable a positive pressure to be applied to thechambers through pins 11034 inserted into holes 11030 and 11032,respectively. A positive pressure can thus simultaneously be applied toall wells within a row, obviating the need to utilize separateconnecting devices to each well.

In operation, solutions are pipetted into the wells 11012, and then chip11008 is placed into bottom portion 11002 of holder 11000. The topholder portion 11014 is placed over chip 11008, and is pressed down byscrews. Raised annular rings 11020 and 11022 on the lower surface of topholder portion 11014 make a seal with the upper surface of the chipwhere the wells are located. Solutions within the wells are exposed topositive pressures within the chamber, and are thereby pushed into theactive area of microfluidic device.

The downward pressure exerted by the chip holder may also pose theadvantage of preventing delamination of the chip from the substrateduring loading. This prevention of delamination may enable the use ofhigher priming pressures.

The chip holder shown in FIG. 33 represents only one possible embodimentof a structure in accordance with the present invention.

Exemplary Systems for Delivery Fluids to Reaction Sites

The positive-displacement cross-injection metering scheme allows forsequential injection of precise sample aliquots from a singlemicrofluidic channel into an array of reaction chambers through apositive displacement cross-injection (PCI) junction. FIGS. 8A-D showsimplified schematic views of positive displacement cross-injection(PCI) for robust and programmable high precision dispensing on chip.

FIG. 8A shows a schematic view of a four port PCI junction. As shown inFIG. 41A, the PCI junction 6200 is formed by the combination of athree-valve peristaltic pump 6202 and a novel four-port cross-injectionjunction with integrated valves on each port. At each junction, two setsof valves 6204 and 6206 are actuated to direct the flow eitherhorizontally or vertically. The split channel architecture creates alarger volume injector region, thereby allowing for an increased numberof injections before recharging.

FIG. 8B shows charging the injector region of the PCI junction. Toexecute the metering task, the flow is switched vertically through thejunction, charging the cross-injector with the sample fluid. Junctionvalves are actuated to direct the flow vertically through the junction,filling the injector region.

FIG. 8C shows precise positive displacement metering by actuation ofperistaltic pump valves in pumping sequence. The flow is then directedhorizontally through the junction and the three valves forming theperistaltic pump are actuated in a five state sequence to advance thefluid in the horizontal direction.

FIG. 8D shows the PCI junction sequentially charged with differentsolutions to create complex multi-component mixtures. Each cycle of theperistaltic pump injects a well-defined volume of sample (approximately80 pL), determined by the dead volume under the middle valve of theperistaltic pump. The deflection of the valve membranes when notactuated is determined by the pressure difference across the membrane.The volume injected during each cycle therefore may be tunedcontinuously, allowing for variable positive displacement metering. Byrepeating the injection sequence, the volume of injected solution may beincreased in 80 pL increments, allowing for the dynamic quantizedcontrol of the final downstream sample concentration.

EXAMPLES Example 1 Fabrication and Operation of Carry-on Reaction Cells

Reaction cells are fabricated in 100 mL (FIG. 9A), 10 mL (FIG. 9B), and1.5 mL (FIG. 9C) volumes by multilayer soft lithography. A reactionchamber 100 is prepared on a first spin layer of varying thicknesses.For a 100 mL reaction chamber, a 100 um recess is patterned with SU8photoresist and a first polydimethylsiloxane (PDMS) elastomeric layer isprepared by spin coating the resist pattern to define a 100 nL reactionchamber (100) with an open side and a closed side. Once the first layeris cured, a via (130) is laser punched through the closed side of thereaction chamber. A second elastomeric layer is prepared by spin coatinga resist pattern to define a 10 um rounded reagent slug channel (150) influidic communication with a 30 um rounded reagent input flow channel(110). The channels are formed as recesses in the second layer. Thereagent input flow channel is formed as a bus channel to connect withadditional reagent slug flow channels when forming multiple reactioncells. When the second layer is cured, via (140) is laser punchedthrough the ceiling of one end of the reagent slug flow channel. A thirdelastomeric layer is prepared as a pour layer over a photoresist mold.The photoresist pattern defines recesses for a first 28 um controlchannel (160) and a second 28 um control channel (170). The controlchannels have a widened recess area that is intended to overlie theceiling membranes of the flow channels for which they are intended tocontrol. When the control channel is sufficiently pressurized, theceiling membrane of the flow channel that is beneath the widened controlchannel recess area will be deflected into the flow channel beneath itthus sealing off the flow channel. When pressure is reduced or removed,the ceiling membrane of the flow channel will deflect upward to reopenthe flow channel. A narrow portion of the control channel carries thecontrol fluid for pressurizing the control channel. By selecting propercontrol channel geometries and flow channel ceiling membranethicknesses, the narrow portion of the control channel overlies portionsof flow channels but does not deflect the flow channels ceiling membraneinto the flow channel at these intersections upon pressurization of thecontrol channel. Also defined in the third elastomeric layer is recessfor a sample inlet channel (120). Once the third elastomeric layer iscured, it is removed from the photoresist mold. The layers are thenaligned and assembled—first by assembling the “third” and “second”layers, and then by assembling the “third/second” layer with the“first”. The elastomeric layers are bonded together by first plasmatreating the surfaces of the layers and then contacting the layers. Thelayers are aligned such that: 1) the first layer is placed with thereaction chamber opening downward; 2) second elastomeric layer isaligned on top of the first layer so that the reagent slug channelrecess (150) is in fluid communication with the slug to reaction chambervia (130); and 3) the third elastomeric layer is aligned so that therecess defining the sample input channel (140) is in fluid communicationwith the sample to slug via (140). The third elastomeric layer is alsoaligned so that the recesses that define control channel 1 (160) with awidened control channel recess area overlies two ends of the reagentslug flow channel. The widened control channel recess area of controlchannel 2 (170) is aligned so that it overlies a portion of the reagentslug flow channel that interfaces to the reagent input flow channel(110). The assembled elastomeric layers form a microfluidic reactioncell that is bonded to a silicon base layer. In this example, the baselayer is a solid monolithic slab of silicon that seals the open end ofthe reaction chamber and also functions as a heat transfer surface fortemperature control of reactions such as polymerase chain reaction(PCR). Reaction chamber volumes of 10 nl and 1.5 nl are prepared with 60um recess in the first elastomeric layer that defines the reactionchamber depth and with reduced length and width dimensions.

To operate the carry-slug reaction cell, control channel 1 ispressurized to deflect the elastomeric membrane valve and close the slugreagent flow channel at its interface with the sample to slug via andthe slug to reaction chamber via. The reagent input flow channel ispressurized and the entire slug reagent flow channel is blind filledwith the desired reagent. Simultaneously, the sample input flow channelis also pressurized and the entire sample flow channel is blind filledup to the valve delineated by control channel 1. Control channel 2 isthen pressurized to deflect the elastomeric membrane valve that closesthe slug reagent flow channel near its interface with the reagent inputflow channel. The sample input flow channel is then re-pressurized andcontrol channel 1 is depressurized to open the slug reagent flow channelat its via connections. The contents of the slug reagent flow channelare then transferred into the reaction chamber through blind filling andunder pressure from the sample input flow channel. The volume of thereaction chamber is in excess of the volume of the slug reagent flowchannel which allows for sample to continue flowing from the sampleinput flow channel and the slug reagent flow channel and to fill up thereaction chamber in the amount that is the difference of the reactionchamber volume and the slug reagent flow channel volume. Control channel1 is repressurized to close off the reaction chamber. The reagent andsample are retained in a mixed solution in the reaction chamber and thereaction is allowed to proceed.

Example 2 Fabrication of a Matrix Reaction Array

A 32×32 elastomeric microfluidic matrix reaction array with slug mixingwas constructed and mixing efficiency was compared to a conventional32×32 elastomeric microfluidic matrix reaction array constructed with asample/reaction chamber adjacent to a reagent chamber and separated byan interface valve.

The matrix reaction array was constructed with each reaction cellcomprising a central 50 nL reaction chamber (400) and a 5 nL slugchannel (250) as shown in FIG. 10. The unit reaction cell of FIG. 2 wasrepeated to prepare a 32×32 matrix fluidic circuit. Using multilayersoft lithography, a first pour layer was patterned with SU8 photoresistto form a mold and then cast with PDMS. The features of the pour layerincluded a 350 um tall reaction chamber and a 30 um tall slug channel.The other, feature, a connecting channel (300) was constructed with aheight of 10 um. A second spin layer was prepared with 15 um tallfeatures for sample input channel (220) and control channel 1 (260) andcontrol channel 2 (270). The second layer was laser punched to form thesample-to-slug via (240). The first pour layer was aligned and bonded tothe second spin layer and the bonded assembly then removed from theresist pattern. The bonded assembly comprising layers 1 and 2 was thenbonded to a thin spun base layer to complete the elastomeric assembly.

FIG. 11 depicts a representative portion of the 32×32 matrix fluidiccircuit. The matrix circuit was divided by column (eg C1, C2, C3, C4)and rows (eg R1, R2, R3, R4). The slug channels 250 of cells in aparticular column, such as column C4, are in fluidic communication viaconnecting channels (300). Connecting channel 300 constitutes a fillinlet for the slug channel. A valve defined by a deflectable membrane ofcontrol channel 2 (270) can fluidically isolate the slug channels of theindividual unit cells of a given column. The isolation of the slugchannels from each other is accomplished by pressurization of controlchannel 2 causing the deflectable membrane portions to deflect into theconnecting channels and sealing off flow through the connectingchannels. The slug channels of a given column are thereforeinterconnected in their native state and capable of being isolated uponactuation of control channel 2. A similar arrangement of interconnectsexists for the rows of the matrix device. A common bus line, sampleinput channel (220), exists in the second layer of the device. This busline provides a common sample input for all of the unit reaction cellsof a particular row of the device. The sample input channel is connectedto the individual slug channels through the sample-to-slug viaconnecting the sample input channel in the second layer to the first endof the slug channel in the first layer. The second end of the slugchannel connects directly to the reaction well (400). The configurationof the slug channel allows the first end to be fluidically isolated fromthe sample-to-slug via and the second end to be isolated from thereaction well with the actuation the valves formed from controlchannel 1. The pressurization of control channel 1, which resides in thesecond layer, causes the deflection of a membrane in the top of thecontrol channel to deflect into slug channel near the first end and thesecond end to fluidically isolate the ends of the slug channel.

Example 3 Demonstration of the Filling and Mixing of a 32×32 MatrixReaction Array

Operation of the matrix reaction array was performed by the followingsteps. Control channel 1 (260) was pressurized to close the valves thatfluidically isolate the ends of the slug channel (FIG. 12A). A yellowfood dye was introduced under pressure through the connecting channels(300) and the slug channels were blind-filled (FIG. 12B). This stepsimulates the filling of the slug channel with reagent. Following thefilling of the slug channels, control channel 2 (270) is pressurized toactuate the valves that close off the connecting channels (300) andthereby isolate the individual slug channels from the other slugchannels in the columns. Although all columns in the reaction array and,accordingly, all slug channels, are filled with the same yellow food dyein this example, there is no interconnection between the connectingchannels and the slug channels of the individual columns. Following theblind filling of the slug channels and their isolation, a blue food dyewas introduced under pressure into all sample input channels (220) (FIG.12D). The control channels 1 were then depressurized to open theinterface valves that were previously closed to isolate the ends of theslug channels (FIG. 12E). The blue food dye that represents the samplein this Example, enters the slug channel at the first end and pushes thereagent into the reaction well (FIG. 12F). This resulted in a highlymixed, loaded reaction well (400) containing the 5 nL of yellow food dyereagent surrogate and 45 nL of blue food dye sample surrogate (50 nLtotal reaction well volume) (FIG. 12G). Finally, in this demonstration,control channels 1 are pressurized which results in the closure of theinterface valves (FIG. 12H). Although all rows in the reaction arrayand, accordingly, all sample input channels, are filled with the sameblue food dye in this example, there is no interconnection between thesample input channels of the individual rows and different samples canbe introduced into the individual rows. In the configuration of thisExample, 32 separate samples can be simultaneously mixed and loaded intoreaction wells with 32 separate reagents for 1024 individualexperiments.

Example 4 Flow Channel Provided with Multiple Mixing Segments

FIG. 13 is a representation of a flow channel that provides for multipleslug mixing segments. Flow channel (510) has a first end gated by valve515 and a second end gated by valve 555 that opens into reaction well(580). The reaction well has an optional outlet channel (560) that isgated by valve 565. Along the length of the flow channel are multiplejunction inlets: 520, 530, 540, and 550, each gated respectively byvalves 518, 528, 538, and 548. Each slug mixing segment is defined by avalve pair that brackets the inlet junction. A first slug mixing segmentis defined by the segment of the flow channel defined by inlet channel520 and valves 515 and 525. A second slug mixing segment is defined byinlet channel 530 and valves 525 and 535. A third slug mixing segment isdefined by inlet channel 540 and valves 535 and 545. A fourth slugmixing segment is defined by inlet channel 550 and valves 545 and 555.In this type of arrangement, multiple solutions may be introduced intothe flow channel by blind filing against the valves that define theirrespective segments. Their inlet junction valves are then closed, thesegment valves are opened and the slugs are pushed into the reactionchamber by flow of a solution through the flow channel (510) to yield awell mixed solution. It is not necessary that all segments of the flowchannel are filled with a solution before the carry-on mixing takesplace. This gives flexibility in what reagents are used in a particularreaction.

Having described several embodiments, it will be recognized by those ofskill in the art that various modifications, alternative constructions,and equivalents may be used without departing from the spirit of theinvention. Additionally, a number of well known processes and elementshave not been described in order to avoid unnecessarily obscuring thepresent invention. Accordingly, the above description should not betaken as limiting the scope of the invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed.The upper and lower limits of these smaller ranges may independently beincluded or excluded in the range, and each range where either, neitheror both limits are included in the smaller ranges is also encompassedwithin the invention, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a process” includes aplurality of such processes and reference to “the electrode” includesreference to one or more electrodes and equivalents thereof known tothose skilled in the art, and so forth.

Also, the words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, acts, orgroups.

What is claimed is:
 1. A positive-displacement cross-injection junctionin an elastomeric microfluidic device, the positive-displacementcross-injection junction comprising: a split channel forming a loop; afirst flow channel intersecting the split channel; a peristaltic pumpcomprising first, second, and third valves, the peristaltic pumppositioned to meter fluid through the first flow channel into the splitchannel; a first cross channel that joins the first flow channel betweenthe peristaltic pump and the split channel; a fourth valve positioned toopen and close the first cross channel; a second flow channel that joinsthe split channel approximately opposite the first flow channel; a fifthvalve positioned to open and close the second flow channel; a secondcross channel that joins the second flow channel between the splitchannel and the fifth valve; a sixth valve positioned to open and closethe second cross channel.
 2. A slug mixing arrangement in an elastomericmicrofluidic device, the slug mixing arrangement comprising: a reactionwell; a flow channel in fluid communication with the reaction well atone end of the flow channel and in fluid communication with a supply ofa solution at a second end of the flow channel; at least three valvesdividing the flow channel into segments, one of the valves positioned toopen and close the flow channel at its connection to the reaction well,and another of the valves positioned to open and close the flow channelat its connection to the supply of solution, the remaining valvesdividing the flow channel into segments between the first and secondvalves; at least two junction inlets, one junction inlet for eachsegment of the flow channel between the first and second valves, eachjunction inlet in fluid communication with its respective segment of theflow channel and gated by a respective valve.